Adenosine A1 receptor-mediated inhibition of in vitro prolactin secretion from the rat anterior pituitary

In previous studies, we demonstrated biphasic purinergic effects on prolactin (PRL) secretion stimulated by an adenosine A2 agonist. In the present study, we investigated the role of the activation of adenosine A1 receptors by (R)-N6-(2-phenylisopropyl)adenosine (R-PIA) at the pituitary level in in vitro PRL secretion. Hemipituitaries (one per cuvette in five replicates) from adult male rats were incubated. Administration of R-PIA (0.001, 0.01, 0.1, 1, and 10 µM) induced a reduction of PRL secretion into the medium in a U-shaped dose-response curve. The maximal reduction was obtained with 0.1 µM R-PIA (mean ± SEM, 36.01 ± 5.53 ng/mg tissue weight (t.w.)) treatment compared to control (264.56 ± 15.46 ng/mg t.w.). R-PIA inhibition (0.01 µM = 141.97 ± 15.79 vs control = 244.77 ± 13.79 ng/mg t.w.) of PRL release was blocked by 1 µM cyclopentyltheophylline, a specific A1 receptor antagonist (1 µM = 212.360 ± 26.560 ng/mg t.w.), whereas cyclopentyltheophylline alone (0.01, 0.1, 1 µM) had no effect. R-PIA (0.001, 0.01, 0.1, 1 µM) produced inhibition of PRL secretion stimulated by both phospholipase C (0.5 IU/mL; 977.44 ± 76.17 ng/mg t.w.) and dibutyryl cAMP (1 mM; 415.93 ± 37.66 ng/mg t.w.) with nadir established at the dose of 0.1 µM (225.55 ± 71.42 and 201.9 ± 19.08 ng/mg t.w., respectively). Similarly, R-PIA (0.01 µM) decreased (242.00 ± 24.00 ng/mg t.w.) the PRL secretion stimulated by cholera toxin (0.5 mg/mL; 1050.00 ± 70.00 ng/mg t.w.). In contrast, R-PIA had no effect (468.00 ± 34.00 ng/mg t.w.) on PRL secretion stimulation by pertussis toxin (0.5 mg/mL; 430.00 ± 26.00 ng/mg t.w.). These results suggest that inhibition of PRL secretion after A1 receptor activation by R-PIA is mediated by a Gi protein-dependent mechanism.

Development of fetal nicotine and muscarinic receptors in utero

The role of acetylcholine in the central and peripheral nervous systems is well established in adults. Cholinergic modulation of vascular functions and body fluid balance has been extensively studied. In the embryo-fetus, cholinergic receptors are widespread in the peripheral and central systems, including smooth muscle and the epithelial lining of the cardiovascular, digestive, and urinary systems, as well as in the brain. Fetal nicotine and muscarinic receptors develop in a pattern (e.g., amount and distribution) related to gestational periods. Cholinergic mechanisms have been found to be relatively intact and functional in the control of vascular homeostasis during fetal life in utero at least during the last third of gestation. This review focuses on the development of fetal nicotine and muscarinic receptors, and provides information indicating that central cholinergic systems are well developed in the control of fetal blood pressure and body fluid balance before birth. Therefore, the development of cholinergic systems in utero plays an important role in fetal vascular regulation, gastrointestinal motility, and urinary control.

Effect of 5-HT1B receptor agonists injected into the prefrontal cortex on maternal aggression in rats

Serotonin (5-HT1B) receptors play an essential role in the inhibition of aggressive behavior in rodents. CP-94,253, a 5-HT1B receptor agonist, can reduce aggression in male mice when administered directly into the ventro-orbitofrontal (VO) prefrontal cortex (PFC). The objective of the current study was to assess the effects of two selective 5-HT1B receptor agonists (CP-94,253 and CP-93,129), microinjected into the VO PFC, on maternal aggressive behavior after social instigation in rats. CP-94,253 (0.56 µg/0.2 µL, N = 8, and 1.0 µg/0.2 µL, N = 8) or CP-93,129 (1.0 µg/0.2 µL, N = 9) was microinjected into the VO PFC of Wistar rats on the 9th day postpartum and 15 min thereafter the aggressive behavior by the resident female against a male intruder was recorded for 10 min. The frequency and duration of aggressive and non-aggressive behaviors were analyzed using ANOVA and post hoc tests. CP-93,129 significantly decreased maternal aggression. The frequency of lateral attacks, bites and pinnings was reduced compared to control, while the non-aggressive behaviors and maternal care were largely unaffected by this treatment. CP-94,253 had no significant effects on aggressive or non-aggressive behaviors when microinjected into the same area of female rats. CP-93,129, a specific 5-HT1B receptor agonist, administered into the VO PFC reduced maternal aggressive behavior, while the CP-94,253 agonist did not significantly affect this behavior after social instigation in female rats. We conclude that only the 5-HT1B receptor agonist CP-93,129 administered into the VO PFC decreased aggression in female rats postpartum after social instigation.

Effects of pentoxifylline treatment before freezing on motility, viability and acrosome status of poor quality human spermatozoa cryopreserved by the liquid nitrogen vapor method

The objective of the present study was to investigate the effects of the direct addition of pentoxifylline (PF) to the ejaculates of men with poor sperm quality before freezing on post-thaw sperm motility, viability, acrosome integrity, and agonist-induced acrosome reaction. Semen specimens from 16 infertile men with impaired sperm count and motility (oligoasthenozoospermia) were divided into two equal aliquots: one received no treatment (control) while the other was incubated with 5 mM PF (treated). Both aliquots were cryopreserved by the liquid nitrogen vapor method. Motility was assessed according to WHO criteria. Acrosome integrity and spontaneous and calcium ionophore-induced acrosome reactions were assessed with fluorescein isothiocyanate-conjugated peanut agglutinin combined with a supra-vital dye (Hoechst-33258). Cryopreservation impaired sperm motility (percentage reduction: 87.4 (interquartile range, IQ: 70.3-92.9) vs 89.1 (IQ: 72.7-96.0%)), viability (25.9 (IQ: 22.2-29.7) vs 25.6 (IQ: 19.7-40.3%)) and acrosome integrity (18.9 (IQ: 5.4-38.9) vs 26.8 (IQ: 0.0-45.2%)) to the same extent in both treated and control aliquots. However, PF treatment before freezing improved the acrosome reaction to ionophore challenge test scores in cryopreserved spermatozoa (9.7 (IQ: 6.6-19.7) vs 4.8 (IQ: 0.5-6.8%); P = 0.002). These data show that pre-freeze treatment of poor quality human sperm with pentoxifylline did not improve post-thaw motility or viability nor did it prevent acrosomal loss during the freeze-thaw process. However, PF, as used, improved the ability of thawed spermatozoa to undergo the acrosome reaction in response to calcium ionophore. The present data indicate that treatment of poor quality human sperm with PF may enhance post-thaw sperm fertilizing ability.

Effect of 4-aminoantipyrine on gastric compliance and liquid emptying in rats

Dipyrone (Dp) delays gastric emptying (GE) in rats. There is no information about whether 4-aminoantipyrine (AA), one of its metabolites, has the same effect. The objectives of the present study were to assess the effects of AA and Dp on GE when administered intravenously (iv) and intracerebroventricularly (icv) (240 µmol/kg and 4 µmol/animal, respectively) and on gastric compliance when administered iv (240 µmol/kg). GE was determined in male Wistar rats weighing 250-300 g (5-10 per group) after icv or iv injection of the drug by measuring percent gastric retention (GR) of a saline meal labeled with phenol red 10 min after administration by gavage. Gastric compliance was estimated in anesthetized rats (10-11 per group), with the construction of volume-pressure curves during intragastric infusion of a saline meal. Compliance was significantly greater in animals receiving Dp (mean ± SEM = 0.26 ± 0.009 mL/mmHg) and AA (0.24 ± 0.012 mL/mmHg) than in controls (0.19 ± 0.009 mL/mmHg). AA and Dp administered iv significantly increased GR (64.4 ± 2.5 and 54.3 ± 3.8%, respectively) compared to control (34 ± 2.2%), a phenomenon observed only with Dp after icv administration. Subdiaphragmatic vagotomy reduced the effect of AA (GR = 31.4 ± 1.5%) compared to sham-treated animals. Baclofen, a GABA B receptor agonist, administered icv significantly reduced the effect of AA (GR = 28.1 ± 1.3%). We conclude that Dp and AA increased gastric compliance and AA delayed GE, with the participation of the vagus nerve, through a pathway that does not involve a direct action of the drug on the central nervous system.

Endothelium-dependent and -independent hepatic artery vasodilatation is not impaired in a canine model of liver ischemia-reperfusion injury

We investigated whether hepatic artery endothelium may be the earliest site of injury consequent to liver ischemia and reperfusion. Twenty-four heartworm-free mongrel dogs of either sex exposed to liver ischemia/reperfusion in vivo were randomized into four experimental groups (N = 6): a) control, sham-operated dogs, b) dogs subjected to 60 min of ischemia, c) dogs subjected to 30 min of ischemia and 60 min of reperfusion, and d) animals subjected to 45 min of ischemia and 120 min of reperfusion. The nitric oxide endothelium-dependent relaxation of hepatic artery rings contracted with prostaglandin F2a and exposed to increasing concentrations of acetylcholine, calcium ionophore A23187, sodium fluoride, phospholipase-C, poly-L-arginine, isoproterenol, and sodium nitroprusside was evaluated in organ-chamber experiments. Lipid peroxidation was estimated by malondialdehyde activity in liver tissue samples and by blood lactic dehydrogenase (LDH), serum aspartate aminotransferase (AST) and serum alanine aminotransferase (ALT) activities. No changes were observed in hepatic artery relaxation for any agonist tested. The group subjected to 45 min of ischemia and 120 min of reperfusion presented marked increases of serum aminotransferases (ALT = 2989 ± 1056 U/L and AST = 1268 ± 371 U/L; P < 0.01), LDH = 2887 ± 1213 IU/L; P < 0.01) and malondialdehyde in liver samples (0.360 ± 0.020 nmol/mgPT; P < 0.05). Under the experimental conditions utilized, no abnormal changes in hepatic arterial vasoreactivity were observed: endothelium-dependent and independent hepatic artery vasodilation were not impaired in this canine model of ischemia/reperfusion injury. In contrast to other vital organs and in the ischemia/reperfusion injury environment, dysfunction of the main artery endothelium is not the first site of reperfusion injury.

Glutamate receptors as seen by light: spectroscopic studies of structure-function relationships

Ionotropic glutamate receptors are major excitatory receptors in the central nervous system and also have a far reaching influence in other areas of the body. Their modular nature has allowed for the isolation of the ligand-binding domain and for subsequent structural studies using a variety of spectroscopic techniques. This review will discuss the role of specific ligand:protein interactions in mediating activation in the a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid subtype of glutamate receptors as established by various spectroscopic investigations of the GluR2 and GluR4 subunits of this receptor. Specifically, this review will provide an introduction to the insight gained from X-ray crystallography and nuclear magnetic resonance investigations and then go on to focus on studies utilizing vibrational spectroscopy and fluorescence resonance energy transfer to study the behavior of the isolated ligand-binding domain in solution and discuss the importance of specific ligand:protein interactions in the mechanism of receptor activation.

Immunohistochemical characterization of the chick marginal retina

The retina is a highly differentiated tissue with a complex layered structure that has been extensively characterized. However, most of the previous studies focused on the histology of the central retina while little is known about the cellular composition, organization and function of the marginal retina. Recent research has identified a subpopulation of multipotential progenitor cells in the marginal regions of the retina, closest to the ciliary body ("ciliary marginal zone"). These cells are capable of differentiation in response to an appropriate stimulus. Thus, it is possible that the structure and composition of the marginal retina are distinct from those of the central retina to accommodate the potential addition of newly formed neurons. To characterize the cellular profile of the chick marginal retina, we labeled it immunohistochemically for markers whose staining pattern is well established in the central retina: calbindin, calretinin, protein kinase C, and choline acetyltransferase. Calbindin was present at very low levels in the marginal retina putative photoreceptor layer. Calretinin-positive horizontal cells were also sparse close to the ciliary marginal zone. The bipolar cells in the marginal outer plexiform layer were positive for anti-protein kinase C antibodies, but the density of labeling was also decreased in relation to the central retina. In contrast, the marginal starburst cholinergic amacrine cell pattern was very similar to the central retina. From these data we conclude that the structure of the marginal retina is significantly different from that of the central retina. In particular, the expression of late retina markers in the marginal retina decreased in comparison to the central retina.

Purinergic signalling: past, present and future

The discovery of non-adrenergic, non-cholinergic neurotransmission in the gut and bladder in the early 1960's is described as well as the identification of adenosine 5'-triphosphate (ATP) as a transmitter in these nerves in the early 1970's. The concept of purinergic cotransmission was formulated in 1976 and it is now recognized that ATP is a cotransmitter in all nerves in the peripheral and central nervous systems. Two families of receptors to purines were recognized in 1978, P1 (adenosine) receptors and P2 receptors sensitive to ATP and adenosine diphosphate (ADP). Cloning of these receptors in the early 1990's was a turning point in the acceptance of the purinergic signalling hypothesis and there are currently 4 subtypes of P1 receptors, 7 subtypes of P2X ion channel receptors and 8 subtypes of G protein-coupled receptors. Both short-term purinergic signalling in neurotransmission, neuromodulation and neurosecretion and long-term (trophic) purinergic signalling of cell proliferation, differentiation, motility, death in development and regeneration are recognized. There is now much known about the mechanisms underlying ATP release and extracellular breakdown by ecto-nucleotidases. The recent emphasis on purinergic neuropathology is discussed, including changes in purinergic cotransmission in development and ageing and in bladder diseases and hypertension. The involvement of neuron-glial cell interactions in various diseases of the central nervous system, including neuropathic pain, trauma and ischemia, neurodegenerative diseases, neuropsychiatric disorders and epilepsy are also considered.

Unconventional neurotransmitters, neurodegeneration and neuroprotection

Neurotransmitters are also involved in functions other than conventional signal transfer between nerve cells, such as development, plasticity, neurodegeneration, and neuroprotection. For example, there is a considerable amount of data indicating developmental roles for the glutamatergic, cholinergic, dopaminergic, GABA-ergic, and ATP/adenosine systems. In this review, we discuss the existing literature on these "new" functions of neurotransmitters in relation to some unconventional neurotransmitters, such as the endocannabinoids and nitric oxide. Data indicating both transcriptional and post-transcriptional modulation of endocannabinoid and nitrinergic systems after neural lesions are discussed in relation to the non-conventional roles of these neurotransmitters. Knowledge of the roles of neurotransmitters in brain functions other than information transfer is critical for a more complete understanding of the functional organization of the brain and to provide more opportunities for the development of therapeutical tools aimed at minimizing neuronal death.

Glutamate-N-methyl-D-aspartate receptor modulation and minocycline for the treatment of patients with schizophrenia: an update

Growing consistent evidence indicates that hypofunction of N-methyl-D-aspartate (NMDA) transmission plays a pivotal role in the neuropathophysiology of schizophrenia. Hence, drugs which modulate NMDA neurotransmission are promising approaches to the treatment of schizophrenia. The aim of this article is to review clinical trials with novel compounds acting on the NMDA receptor (NMDA-R). This review also includes a discussion and translation of neuroscience into schizophrenia therapeutics. Although the precise mechanism of action of minocycline in the brain remains unclear, there is evidence that it blocks the neurotoxicity of NMDA antagonists and may exert a differential effect on NMDA signaling pathways. We, therefore, hypothesize that the effects of minocycline on the brain may be partially modulated by the NMDA-R or related mechanisms. Thus, we have included a review of minocycline neuroscience. The search was performed in the PubMed, Web of Science, SciELO, and Lilacs databases. The results of glycine and D-cycloserine trials were conflicting regarding effectiveness on the negative and cognitive symptoms of schizophrenia. D-serine and D-alanine showed a potential effect on negative symptoms and on cognitive deficits. Sarcosine data indicated a considerable improvement as adjunctive therapy. Finally, minocycline add-on treatment appears to be effective on a broad range of psychopathology in patients with schizophrenia. The differential modulation of NMDA-R neurosystems, in particular synaptic versus extrasynaptic NMDA-R activation and specific subtypes of NMDA-R, may be the key mediators of neurogenesis and neuroprotection. Thus, psychotropics modulating NMDA-R neurotransmission may represent future monotherapy or add-on treatment strategies in the treatment of schizophrenia.

Purine nucleotides reduce superoxide production by nitric oxide synthase in a murine sepsis model

Sepsis involves a systemic inflammatory response of multiple endogenous mediators, resulting in many of the injurious and sometimes fatal physiological symptoms of the disease. This systemic activation leads to a compromised vascular response and endothelial dysfunction. Purine nucleotides interact with purinoceptors and initiate a variety of physiological processes that play an important role in maintaining cardiovascular function. The purpose of the present study was to investigate the effects of ATP on vascular function in a lipopolysaccharide (LPS) model of sepsis. LPS induced a significant increase in aortic superoxide production 16 h after injection. Addition of ATP to the organ bath incubation solution reduced superoxide production by the aortas of endotoxemic animals. Reactive Blue, an antagonist of the P2Y receptor, blocked the effect of ATP on superoxide production, and the nonselective P2Y agonist MeSATP inhibited superoxide production. Nitric oxide synthase (NOS) inhibition by L-NAME blocked vascular relaxation and reduced superoxide production in LPS-treated animals. In the presence of L-NAME there was no ATP effect on superoxide production. A vascular reactivity study showed that ATP increased maximal relaxation in LPS-treated animals compared to controls. The presence of ATP induced increases in Akt and endothelial NOS phosphorylated proteins in the aorta of septic animals. ATP reduces superoxide release resulting in an improved vasorelaxant response. Sepsis may uncouple NOS to produce superoxide. We showed that ATP through Akt pathway phosphorylated endothelial NOS and “re-couples” NOS function.

Protective mechanism of agmatine pretreatment on RGC-5 cells injured by oxidative stress

Agmatine has neuroprotective effects on retinal ganglion cells (RGCs) as well as cortical and spinal neurons. It protects RGCs from oxidative stress even when it is not present at the time of injury. As agmatine has high affinity for various cellular receptors, we assessed protective mechanisms of agmatine using transformed RGCs (RGC-5 cell line). Differentiated RGC-5 cells were pretreated with 100 μM agmatine and consecutively exposed to 1.0 mM hydrogen peroxide (H2O2). Cell viability was determined by measuring lactate dehydrogenase (LDH), and the effects of selective alpha 2-adrenergic receptor antagonist yohimbine (0-500 nM) and N-methyl-D-aspartic acid (NMDA) receptor agonist NMDA (0-100 µM) were evaluated. Agmatine’s protective effect was compared to a selective NMDA receptor antagonist MK-801. After a 16-h exposure to H2O2, the LDH assay showed cell loss greater than 50%, which was reduced to about 30% when agmatine was pretreated before injury. Yohimbine almost completely inhibited agmatine’s protective effect, but NMDA did not. In addition, MK-801 (0-100 µM) did not significantly attenuate the H2O2-induced cytotoxicity. Our results suggest that neuroprotective effects of agmatine on RGCs under oxidative stress may be mainly attributed to the alpha 2-adrenergic receptor signaling pathway.

Autonomic control of cardiorespiratory interactions in fish, amphibians and reptiles

Control of the heart rate and cardiorespiratory interactions (CRI) is predominantly parasympathetic in all jawed vertebrates, with the sympathetic nervous system having some influence in tetrapods. Respiratory sinus arrhythmia (RSA) has been described as a solely mammalian phenomenon but respiration-related beat-to-beat control of the heart has been described in fish and reptiles. Though they are both important, the relative roles of feed-forward central control and peripheral reflexes in generating CRI vary between groups of fishes and probably between other vertebrates. CRI may relate to two locations for the vagal preganglionic neurons (VPN) and in particular cardiac VPN in the brainstem. This has been described in representatives from all vertebrate groups, though the proportion in each location is variable. Air-breathing fishes, amphibians and reptiles breathe discontinuously and the onset of a bout of breathing is characteristically accompanied by an immediate increase in heart rate plus, in the latter two groups, a left-right shunting of blood through the pulmonary circuit. Both the increase in heart rate and opening of a sphincter on the pulmonary artery are due to withdrawal of vagal tone. An increase in heart rate following a meal in snakes is related to withdrawal of vagal tone plus a non-adrenergic-non-cholinergic effect that may be due to humoral factors released by the gut. Histamine is one candidate for this role.

Human monocytes tolerant to LPS retain the ability to phagocytose bacteria and generate reactive oxygen species

Tolerance to lipopolysaccharide (LPS) occurs when animals or cells exposed to LPS become hyporesponsive to a subsequent challenge with LPS. This mechanism is believed to be involved in the down-regulation of cellular responses observed in septic patients. The aim of this investigation was to evaluate LPS-induced monocyte tolerance of healthy volunteers using whole blood. The detection of intracellular IL-6, bacterial phagocytosis and reactive oxygen species (ROS) was determined by flow cytometry, using anti-IL-6-PE, heat-killed Staphylococcus aureus stained with propidium iodide and 2',7'-dichlorofluorescein diacetate, respectively. Monocytes were gated in whole blood by combining FSC and SSC parameters and CD14-positive staining. The exposure to increasing LPS concentrations resulted in lower intracellular concentration of IL-6 in monocytes after challenge. A similar effect was observed with challenge with MALP-2 (a Toll-like receptor (TLR)2/6 agonist) and killed Pseudomonas aeruginosa and S. aureus, but not with flagellin (a TLR5 agonist). LPS conditioning with 15 ng/mL resulted in a 40% reduction of IL-6 in monocytes. In contrast, phagocytosis of P. aeruginosa and S. aureus and induced ROS generation were preserved or increased in tolerant cells. The phenomenon of tolerance involves a complex regulation in which the production of IL-6 was diminished, whereas the bacterial phagocytosis and production of ROS was preserved. Decreased production of proinflammatory cytokines and preserved or increased production of ROS may be an adaptation to control the deleterious effects of inflammation while preserving antimicrobial activity.